Project Summary Limited understanding of the immunological mechanisms underlying protective immunity hampers the development of novel vaccines, particularly against human-restricted enteric bacteria such as Salmonella Typhi. The near exclusive reliance on circulating immune cells, which do not necessarily reflect the responses of ?resident? immune cells present in the gut, is another major limitation. Over the past 25 years, in studies largely supported by this R01, and particularly during the last funding period (2013-2018), the PI reported major advances in our understanding the B, T and innate immune responses elicited by the licensed Ty21a oral typhoid and other candidate attenuated S. Typhi vaccines and, more recently, using specimens obtained from a wild type (wt) S. Typhi human challenge model. The latter studies examined, among other key immune cell types, the induction and activation of memory T (TM) subsets and regulatory T cells (TREG), demonstrating, for the first time, an association between defined multifunctional CD8+ subsets and regulatory T cell (TREG) responses and clinical outcome upon challenge. However, many key questions regarding whether the same T subsets are elicited following Ty21a immunization and are associated with protection from challenge with wt S. Typhi and the induction and persistence of gut and systemic immunity following vaccination remain unexplored. These major gaps should be addressed before we can apply this knowledge to the development of more effective oral vaccines. To directly address these major gaps, we propose to continue and greatly expand our findings during the current funding period by evaluating the hypotheses that the characteristics of CD4 and CD8 T-CMI S. Typhi- specific responses, including TM subsets (e.g., TM, T effector (TEFF), T stem-cell like memory (TSCM), and TREG at baseline and at the early stages after immunization and/or challenge with wt S. Typhi play critical roles in protection from infection and persistence of immunity in humans. To this end we will use unique specimens from adults vaccinated and/or challenged with wt S. Typhi, and gut biopsies from participants vaccinated with Ty21a to: Aim 1. Evaluate the hypothesis that defined T cell subsets with gut-homing potential present at baseline and those elicited during the early stages following Ty21a vaccination and/or wt S. Typhi challenge are associated with robust immunity, protection from clinical disease and persistent immunity. Aim 2. Evaluate the hypothesis that protection from infection following wt S. Typhi challenge and robust effector and memory immunity following vaccination is associated with defined regulatory T cell (TREG) responses and depends on a balanced TEFF / TM / TSCM / and TREG responses at baseline and after immunization. Aim 3. Evaluate the hypothesis that robust terminal ileum (TI) CD4 and CD8 TEFF, TM, and T resident/memory (TRM) responses in the absence of TREG cells is associated with long-term maintenance of mucosal and systemic responses (TEFF, TM, TSCM) after oral immunization of volunteers with the Ty21a typhoid vaccine.